Rationally-designed single-chain meganucleases with non-palindromic recognition sequences

Abstract

Disclosed are rationally-designed, non-naturally-occurring meganucleases in which a pair of enzyme subunits having specificity for different recognition sequence half-sites are joined into a single polypeptide to form a functional heterodimer with a non-palindromic recognition sequence. The invention also relates to methods of producing such meganucleases, and methods of producing recombinant nucleic acids and organisms using such meganucleases.

The invention claimed is: 
     
       1. A recombinant single-chain meganuclease comprising:
 a first LAGLIDADG (SEQ ID NO: 55) subunit comprising a polypeptide sequence having at least 85% sequence identity to residues 9-151 of a wild-type I-CreI meganuclease of SEQ ID NO: 1, said first LAGLIDADG (SEQ ID NO: 55) subunit having a first recognition half-site; 
 a second LAGLIDADG (SEQ ID NO: 55) subunit comprising a polypeptide sequence having at least 85% sequence identity to residues 9-151 of a wild-type I-CreI meganuclease of SEQ ID NO: 1, said second LAGLIDADG (SEQ ID NO: 55) subunit having a second recognition half-site; 
 wherein said first and second LAGLIDADG (SEQ ID NO: 55) subunits are covalently joined by a polypeptide linker such that said first LAGLIDADG (SEQ ID NO: 55) domain is N-terminal to said linker and said second LAGLIDADG (SEQ ID NO: 55) domain is C-terminal to said linker; wherein said polypeptide linker comprises 15-56 residues, and 
 wherein said first and second LAGLIDADG (SEQ ID NO: 55) subunits are capable of functioning together to recognize and cleave a non-palindromic DNA sequence which is a hybrid of said first recognition half-site and said second recognition half-site. 
 
     
     
       2. The recombinant single-chain meganuclease of  claim 1  wherein at least one of said LAGLIDADG (SEQ ID NO: 55) domains comprises at least one amino acid modification selected from the group consisting of A26, A28, A32, A40, A44, A46, A70, A79, C24, C28, C32, C33, C38, C40, C44, C46, C68, C70, C75, C79, D32, D33, D44, D46, D70, E26, E30, E32, E33, E38, E40, E42, E44, E46, E68, E70, E75, E77, F33, F68, G46, G70, H139, H28, H32, H33, H38, H46, H68, H70, H75, I24, I32, I33, I38, I40, I44, I79, K24, K26, K28, K30, K32, K38, K44, K46, K66, K68, K70, L32, L33, L38, L44, L68, L70, L75, M66, M68, N30, N32, N38, N44, Q26, Q28, Q30, Q32, Q38, Q40, Q42, Q44, Q46, Q68, Q70, Q75, Q77, R24, R28, R30, R32, R33, R38, R40, R42, R44, R46, R68, R70, R75, R77, S26, S28, S32, S40, S70, S77 T32, T44, T46, V32, V33, V40, V44, V79, Y139, Y33, Y68, and Y75. 
     
     
       3. The recombinant single-chain meganuclease of  claim 1  wherein: each of said LAGLIDADG (SEQ ID NO: 55) subunits has a recognition half-site selected from the group consisting of GAAACTGTC; GACAGTTTC; CAAAACGTC; GACGTTTTG; CAGAACGTC; GACGTTCTG; GGAACTGTC; GACAGTTCC; ATAACGGTC; GACCGTTAT; TTCGCTACC; GGTAGCGAA; TAGGG; CCCTA; TAATGGGAC; GTCCCATTA; GCCGGAAC; GTTCCGGC; AACGGCC; GGCCGTT; TTTACAGA; TCTGTAAA; CTGAGGAGG; and CCTCCTCAG. 
     
     
       4. The recombinant single-chain meganuclease of  claim 1  wherein:
 said polypeptide linker is a flexible linker. 
 
     
     
       5. The recombinant single-chain meganuclease of  claim 4  wherein:
 said linker comprises 15-40 residues. 
 
     
     
       6. The recombinant single-chain meganuclease of  claim 4  wherein:
 said linker comprises 25-31 residues. 
 
     
     
       7. The recombinant single-chain meganuclease of  claim 4  wherein:
 at least 50% of said linker comprises polar uncharged residues. 
 
     
     
       8. The recombinant single-chain meganuclease of  claim 1  wherein:
 said polypeptide linker has a stable secondary structure. 
 
     
     
       9. The recombinant single-chain meganuclease of  claim 8  wherein:
 said stable secondary structure comprises at least two α-helix structures. 
 
     
     
       10. The recombinant single-chain meganuclease of  claim 8  wherein:
 said stable secondary structure comprises from N-terminus to C-terminus a first loop, a first α-helix, a first turn, a second α-helix, and a second loop. 
 
     
     
       11. The recombinant single-chain meganuclease of  claim 8  wherein:
 said linker comprises 23-56 residues.